Magnetic-associated Cell Sorting Research Articles

Abstract B008: Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance in prostate cancer

Abstract Introduction and Objective Several studies have shown that cancer-associated fibroblasts (CAFs), the most abundant component in prostate cancer (PCa) microenvironment, promote resistance to androgen deprivation therapies, and metastatic development. The aim of this study is to elucidate the mechanisms of CAF-induced therapy resistance through the establishment of in vitro 3D co-cultures. Methods In this study, the androgen-dependent PNPCa patient-derived xenograft (PDX) model (derived from soft tissue metastasis), and the androgen-independent LAPC9 PDX model (bone metastasis) are used. Epithelial and stromal cells (human and mouse origin, respectively) are separated through magnetic-associated cell sorting to generate PDX-derived organoids and fibroblasts. Fibroblast RNA and protein expression is assessed through RT-qPCR, Western Blot, and Immunofluorescence. Direct 3D co-cultures are obtained by combining fluorescently labelled organoids and fibroblasts in ultra-low attachment plates in defined media. In indirect co-cultures, organoids are cultured in Spherical Plates (Kugelmeiers) with fibroblasts on transwell inserts. Organoid viability is measured after 9 days by CellTiter-Glo 3D Assay. Results PDX-derived fibroblasts express typical CAF genes (e.g. α-smooth muscle actin and Tenascin C), and androgen receptor (AR), both at RNA and protein level. AR protein expression (as well as RNA expression of the AR target gene Fkbp5) is increased in CAFs upon treatment with dyhydrotestosterone (DHT) 10nM, while co-treatment with the AR inhibitor Enzalutamide 10mM abrogates this effect, suggestive of functional AR signaling. These CAFs can organize into tumor/CAF organoid 3D structures upon combination with PDX-derived tumor epithelial cells, providing an in vitro functional model to study tumor-stromal interactions. In indirect (transwell) co-cultures, CAFs increase the viability of the androgen dependent PNPCa organoids, but not that of androgen independent LAPC9. This growth-promoting effect is observed even upon culturing of PNPCa organoids in non-optimal growth conditions, at low DHT concentrations (DHT 0.5nM and 0.25nM versus the standard DHT 1nM). The factors mediating this process will be identified by gene expression profiling of CAFs and organoids in transwell co-cultures and/or monoculture. Conclusions Our data indicate that PCa metastatic cells modify the properties of neighbor stromal cells to support tumorigenesis, since PDX-derived fibroblasts, originally part of the mouse dermal stroma, display CAF features and active AR signaling. 3D co-cultures represent a useful tool to study the tumor-stromal interactions. Our transwell co-culture experiments suggest that CAFs play an important role in supporting the viability and growth of early metastatic androgen dependent PCa cells (PNPCa) by secreting pro-tumorigenic factors. The possibility that CAFs exert different effects depending on the tumor stage setting (early vs advanced) will be subsequently investigated. Citation Format: Francesco Bonollo, Natalie Sampson, George Thalmann, Sofia Karkampouna, Marianna Kruithof-de Julio. Tumor-stromal 3D co-cultures to study the role of cancer-associated fibroblasts in the acquisition of androgen-deprivation therapy resistance in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B008.

Dystrophin Dp71 Subisoforms Localize to the Mitochondria of Human Cells

Duchenne muscular dystrophy (DMD) is a fatal muscle wasting disease caused by deficiency in dystrophin, a protein product encoded by the DMD gene. Mitochondrial dysfunction is now attracting much attention as a central player in DMD pathology. However, dystrophin has never been explored in human mitochondria. Here, we analyzed dystrophin in cDNAs and mitochondrial fractions of human cells. Mitochondrial fraction was obtained using a magnetic-associated cell sorting (MACS) technology. Dystrophin was analyzed by reverse transcription (RT)-PCR and western blotting using an antibody against the dystrophin C-terminal. In isolated mitochondrial fraction from HEK293 cells, dystrophin was revealed as a band corresponding to Dp71b and Dp71ab subisoforms. Additionally, in mitochondria from HeLa, SH-SY5Y, CCL-136 and HepG2 cells, signals for Dp71b and Dp71ab were revealed as well. Concomitantly, dystrophin mRNAs encoding Dp71b and Dp71ab were disclosed by RT-PCR in these cells. Primary cultured myocytes from three dystrophinopathy patients showed various levels of mitochondrial Dp71 expression. Coherently, levels of mRNA were different in all cells reflecting the protein content, which indicated predominant accumulation of Dp71. Dystrophin was demonstrated to be localized to human mitochondrial fraction, specifically as Dp71 subisoforms. Myocytes derived from dystrophinopathy patients manifested different levels of mitochondrial Dp71, with higher expression revealed in myocytes from Becker muscular dystrophy (BMD) patient-derived myocytes.

Abstract A38: RNA-seq of diagnostic and relapse disseminated tumor cells (DTCs) in stage M neuroblastoma patients and first steps towards DTC-derived xenografts in zebrafish embryos

Abstract Neuroblastoma (NB) is the most common tumor diagnosed in the first year of life. More than 90% of stage M NB patients present with disseminated tumor cells (DTCs) in the bone marrow (BM). Although these cells represent a major obstacle to treatment, their global gene expression has, so far, been only poorly analyzed. GD2POS DTCs of stage M NB patients were separated from the remaining BM-derived nontumor mononuclear cells (MNCs) by applying magnetic associated cell sorting. mRNA of diagnostic DTCs (n=22) and MNCs (n=14), primary tumors (n=16), relapse DTCs (n=20) and MNCs (n=14) was isolated and used for RNA-seq (50bp, single-end) in order to characterize the biology of DTCs. Furthermore, four DTC-derived neuroblastoma cell lines were established and used to generate a robust patient-derived zebrafish xenograft model. Annexin V staining (apoptosis), EdU proliferation assays, and transwell migration assays of the four cell lines were performed in vitro at temperatures between 34°C and 37°C. 322 differently expressed genes (DEG) were identified comparing primary tumors and DTCs (q < 0.001, |log2FC|>2). Especially, genes encoded by mitochondrial DNA (mtDNA) were upregulated in DTCs. Furthermore, we found the gene expression signature of relapse DTCs largely resembling those of diagnostic DTCs, with only 113 DEG under relaxed cut-offs (q < 0.01, |log2FC|>0.5). Notably, relapse DTCs showed a positional enrichment of 31 downregulated genes on chromosome 19, including five tumor-suppressor genes: SIRT6, BBC3/PUMA, STK11, CADM4, and GLTSCR2. Genomic analysis of 34 DTC samples revealed clonal deletions affecting a portion of the 31 downregulated genes encoded by chromosome 19. Although these deletions occurred at a higher frequency in relapse patients (9 out of 17 samples) as compared to the diagnostic samples (4 out of 17 samples), the positional distribution of downregulated genes did not coincide with the deleted ones. While chromosomal deletions in relapse samples were mainly found on 19q (8 out of 20 samples), most of the downregulated genes were encoded by 19p, which rarely contained deletions (3 out of 20 samples). The downregulation of PUMA (p=0.002) and CADM4 (p=0.02) was associated with a worse event-free survival in stage M patients (survival analysis performed in R2 with the Oberthuer dataset). Furthermore, four DTC-derived cell lines were tested for suitability of injecting into zebrafish embryos and subsequent functional testing. In vitro analysis revealed that in two cell lines (STA-NB-8 and STA-NB-12) proliferation and apoptosis were not affected by lower temperatures (34°C), at which zebrafish embryos still can develop properly, while in the remaining two cell lines both were impaired. Fewer STA-NB-15 and STA-NB-19 cells were in the S phase at 34°C (18.6% of STA-NB-15 and 24.2% of STA-NB-19 cells) as compared to 37°C (37.8% of STA-NB-15 and 45.1% of STA-NB-19 cells). Additionally, more of the STA-NB-15 cells were undergoing apoptosis at 34°C (50.3% Annexin V positive cells) as compared to 37°C (33.9% Annexin V positive cells). This first RNA-seq analysis of neuroblastoma DTCs revealed their unique expression profile in contrast to tumors, but showed less pronounced differences between diagnostic and relapse DTCs. The latter preferentially affected the downregulation of genes encoded by chromosome 19, which might be associated with treatment failure and disease progression. The establishment of xenografts in zebrafish embryos with DTC-derived cells will enable in vivo functional studies and optimize the generation of patient-derived xenografts in zebrafish embryos, which will allow drug screening and functional studies for personalized treatment approaches. Citation Format: Fikret Rifatbegovic, Sabine Taschner-Mandl, Martin Distel, M. Reza Abbasi, Christian Frech, Andrea Ziegler, Inge M. Ambros, Peter F. Ambros. RNA-seq of diagnostic and relapse disseminated tumor cells (DTCs) in stage M neuroblastoma patients and first steps towards DTC-derived xenografts in zebrafish embryos [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A38.

Bone marrow-derived lineage-negative cells accelerate skin regeneration in vivo.

Cell-based therapy is a promising strategy for promoting tissue regeneration when conventional treatments are not effective. ehT choice of the accessible source to obtain a sufficient cell amount and the use of suitable biomaterials to improve the cell delivery efficiency are the main tasks for safe, effective, and reliable application of stem cell therapy. In this study, we have compared the influence of bone marrow-derived Lin¯ cells on skin regeneration after local transplantation with or without type I collagen-based gel in a BALB/c mice full-thickness wound model. Lin¯ cells were isolated using magnetic-associated cell sorting and identified by flow cytometry. Cytokine gene expression was examined using real-time PCR. Our results show that the bone marrow-derived Lin¯ cell population demonstrates the properties to stimulate the skin tissue regeneration. Significant accelerated wound closure was revealed after cell transplantation (P < 0.05). Histological analysis indicated the earliest inhibition of inflammation, accelerated reepithelialization, and evenly distributed skin appendages in the neodermis after Lin¯ cell transplantation with type I collagen gel. eTh significant changes in mRNA levels of cytokines TNF-α, IL-10, TGF-β, and VEGF after Lin¯ cell transplantation were confirmed by RT-PCR (P < 0.05). eTh ability to positively control the reactions taking place during the wound healing process gives the advantage to the bone marrow Lin¯ cell population to be used as a cell source for therapy.

RNA sequencing analysis reveals quiescent microglia isolation methods from postnatal mouse brains and limitations of BV2 cells

BackgroundMicroglia play key roles in neuron–glia interaction, neuroinflammation, neural repair, and neurotoxicity. Currently, various microglial in vitro models including primary microglia derived from distinct isolation methods and immortalized microglial cell lines are extensively used. However, the diversity of these existing models raises difficulty in parallel comparison across studies since microglia are sensitive to environmental changes, and thus, different models are likely to show widely varied responses to the same stimuli. To better understand the involvement of microglia in pathophysiological situations, it is critical to establish a reliable microglial model system.MethodsWith postnatal mouse brains, we isolated microglia using three general methods including shaking, mild trypsinization, and CD11b magnetic-associated cell sorting (MACS) and applied RNA sequencing to compare transcriptomes of the isolated cells. Additionally, we generated a genome-wide dataset by RNA sequencing of immortalized BV2 microglial cell line to compare with primary microglia. Furthermore, based on the outcomes of transcriptional analysis, we compared cellular functions between primary microglia and BV2 cells including immune responses to LPS by quantitative RT-PCR and Luminex Multiplex Assay, TGFβ signaling probed by Western blot, and direct migration by chemotaxis assay.ResultsWe found that although the yield and purity of microglia were comparable among the three isolation methods, mild trypsinization drove microglia in a relatively active state, evidenced by high amount of amoeboid microglia, enhanced expression of microglial activation genes, and suppression of microglial quiescent genes. In contrast, CD11b MACS was the most reliable and consistent method, and microglia isolated by this method maintained a relatively resting state. Transcriptional and functional analyses revealed that as compared to primary microglia, BV2 cells remain most of the immune functions such as responses to LPS but showed limited TGFβ signaling and chemotaxis upon chemoattractant C5a.ConclusionsCollectively, we determined the optimal isolation methods for quiescent microglia and characterized the limitations of BV2 cells as an alternative of primary microglia. Considering transcriptional and functional differences, caution should be taken when extrapolating data from various microglial models. In addition, our RNA sequencing database serves as a valuable resource to provide novel insights for appropriate application of microglia as in vitro models.

Senescent T-Cells Promote Bone Loss in Rheumatoid Arthritis.

T-cells are critical players in the pathogenesis of osteoporosis in patients with rheumatoid arthritis (RA). Premature senescence of lymphocytes including the accumulation of senescent CD4+ T-cells is a hallmark feature of RA. Whether T-cell senescence is associated with bone loss in RA patients is elusive so far. This includes a prospective study of consecutive patients with RA (n = 107), patients with primary osteopenia/-porosis (n = 75), and healthy individuals (n = 38). Bone mineral density (BMD) was determined by dual-energy X-ray absorptiometry scan. Flow cytometry, magnetic-associated cell sorting, and cell culture experiments were performed to analyze the pro-osteoclastic phenotype and the function of senescent CD4+CD28- T-cells. Patients with osteopenia/-porosis yielded a higher prevalence of senescent CD4+CD28- T-cells than individuals with normal BMD, in the RA, as well as in the non-RA cohort. Receptor activator of nuclear factor kappa-B ligand (RANKL) was expressed at higher levels on CD4+CD28- T-cells as compared to CD28+ T-cells. Stimulation with interleukin-15 led to an up-regulation of RANKL expression, particularly on CD28- T-cells. CD4+CD28- T-cells induced osteoclastogenesis more efficiently than CD28+ T-cells. Our data indicate that senescent T-cells promote osteoclastogenesis more efficiently than conventional CD28+ T-cells, which might contribute to the pathogenesis of systemic bone loss in RA and primary osteoporosis.

Novel Senescent Regulatory T-Cell Subset with Impaired Suppressive Function in Rheumatoid Arthritis.

Premature senescence of lymphocytes is a hallmark of inflammatory rheumatic diseases such as rheumatoid arthritis (RA). Early T-cell aging affects conventional T-cells but is presumably not limited to this cell population; rather it might also occur in the regulatory T-cells (Tregs) compartment. In RA, Tregs fail to halt aberrant immune reactions and disease progression. Whether this is associated with early Treg senescence leading to phenotypic and functional changes of this subset is elusive so far. Eighty-four RA patients and 75 healthy controls were prospectively enrolled into the study. Flow cytometry, magnetic-associated cell sorting, and cell culture experiments were performed for phenotypic and functional analyses of Treg subsets. T-cell receptor excision circle (TREC) levels and telomere lengths were determined using RT-PCR. In this paper, we describe the novel CD4+FoxP3+CD28- T-cell subset (CD28- Treg-like cells) in RA patients revealing features of both Tregs and senescent T-cells: Treg surface/intracellular markers such as CD25, CTLA-4, and PD-1 as well as FOXP3 were all expressed by CD28- Treg-like cells, and they yielded signs of premature senescence including reduced TREC levels and an accumulation of γH2AX. CD28- Treg-like could be generated in vitro by stimulation of (CD28+) Tregs with TNF-α. CD28- Treg-like cells insufficiently suppressed the proliferation of effector T-cells and yielded a pro-inflammatory cytokine profile. In conclusion, we describe a novel T-cell subset with features of Tregs and senescent non-Tregs. These cells may be linked to an aberrant balance between regulatory and effector functions in RA.

Inhibitory effect of epirubicin-loaded lipid microbubbles with conjugated anti-ABCG2 antibody combined with therapeutic ultrasound on multiple myeloma cancer stem cells

Ultrasound-targeted microbubble destruction (UTMD) technique is thought to improve the chemotherapeutic agent delivery from microbubbles (MBs) in tumor tissues and reduce the side effects in non-tumor tissues. Multiple myeloma (MM) is a bone marrow cancer and remains to be an incurable disease. In this study, we used the UTMD technique to investigate the inhibitory effect of our developed novel reagent on MM cancer stem cells (CD138−CD34−MM CSCs) that are MM cells with CD138−CD34− phenotypes, responsible for MM-initiating potential, drug resistance and eventual relapse. The preparatory steps of novel reagent was first epirubicin (EPI)-loaded in the lipid MBs that was consisted of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]-biotin, dipalmitoyl-phosphatidylglycerol and 25-NBD-cholesterol, then anti-ABCG2 monoclonal antibody (mAb) was conjugated onto the MB surface to form EPI-MBs+mAb. CD138−CD34−MM CSCs were isolated from human MM RPMI 8226 cell line by the magnetic associated cell sorting method. The results showed that the attenuated proliferation, migration and invasion ability, and increased apoptosis were observed when MM CSCs were incubated with a various agents. EPI-MBs+mAb combined with therapeutic ultrasound significantly promoted the MM CSC apoptosis compared with EPI, EPI-MBs alone or EPI-MBs+mAb without ultrasound exposure. These results suggest that the developed EPI-MBs+mAb combined with therapeutic ultrasound remarkably induced MM CSC apoptosis in vitro.

Decrease of ZEB1 expression inhibits the B16F10 cancer stem-like properties.

Increasing evidence supports that cancer stem cells (CSCs) are responsible for driving tumor initiation and maintenance. Zinc-finger E-box binding homeobox 1 (ZEB1) is a transcription factor for regulating tumor progression, and contributes to maintenance of CSC-like properties. The goal of the present study is to investigate the effect of decreasing ZEB1 expression on the B16F10 CSC-like properties. The recombinant shRNA targeting ZEB1 were transfected into melanoma B16F10 cells, and shZEB1-CD133(+)CD44(+) CSCs were isolated from the stable transfected cells using the magnetic-associated cell sorting method. The shZEB1-CD133(+)CD44(+) CSC-like properties were systematically analyzed. The results show the B16F10 shZEB1-CD133(+)CD44(+) CSCs significantly decreased the ability of clonogenicity, cellular proliferation, migration, and invasion. Importantly, tumorigenicity and tumor lung metastasis was significantly inhibited in B16F10 shZEB1-CD133(+)CD44(+) CSCs compared with B16F10 scramble-CD133(+)CD44(+) CSCs. The decrease of ZEB1 expression markedly resulted in down-regulation of vimentin and N-cadherin expression as well as up-regulation of E-cadherin expression in tumor tissues from the mice injected with B16F10 shZEB1-CD44(+)CD133(+) CSCs. These findings contribute to understanding the maintenance of B16F10 CD133(+)CD44(+) CSC-like properties that was closely associated with ZEB1 expression. ZEB1 may serve as a new therapeutic target for treatment of malignant melanoma.

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Fe3O4 nanoparticle loaded paclitaxel induce multiple myeloma apoptosis by cell cycle arrest and increase cleavage of caspases in vitro

Multiple myeloma (MM) still remains an incurable disease in spite of extending the patient survival by new therapies. The hypothesis of cancer stem cells (CSCs) states that although chemotherapy kills most tumor cells, it is believed to leave a reservoir of CSCs that allows the tumor cell propagation. The objective of this research was to evaluate the therapeutic effect of new paclitaxel-Fe3O4 nanoparticles (PTX-NPs) with an average size range of 7.17 ± 1.31 nm on MM CSCs in vitro. The characteristics of CD138−CD34− cells, isolated from human MM RPMI 8226 and NCI-H929 cell lines by the magnetic associated cell sorting method, were identified by the assays of colony formation, cell proliferation, drug resistance, cell migration, and tumorigenicity in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, respectively. Inhibitory effects of PTX-NPs on CD138−CD34− cells were evaluated by a variety of assays in vitro. The results showed that the CD138−CD34− cells were capable of forming colonies, exhibited high proliferative and migratory ability, possessed a strong drug resistance, and had powerful tumorigenicity in NOD/SCID mice compared to non-CD138−CD34− cells. PTX-NPs significantly inhibited CD138− CD34− cell viability and invasive ability, and resulted in G0/G1 cell cycle arrest and apoptosis compared with PTX alone. We concluded that the CD138−CD34− phenotype cells might be CSCs in RPMI 8226 and NCI-H929 cell lines. PTX-NPs had an obvious inhibitory effect on MM CD138−CD34− CSCs. The findings may provide a guideline for PTX-NPs’ treatment of MM CSCs in preclinical investigation.

CD44 as a molecular marker to screen cancer stem cells in hypopharyngeal cancer

Conclusions: The CD44+ cells have a stronger proliferative capacity and higher tumorigenic potential than the CD44– cells, which suggests that the cancer stem cells of hypopharyngeal cancer may exist in the CD44+ tumor cell population. Therefore, we propose that CD44 is an important biological marker to screen cancer stem cells of hypopharyngeal cancer. Objectives: To study the significance of CD44 as a molecular marker for screening cancer stem cells in hypopharyngeal cancer. Methods: The CD44 expression levels in the hypopharyngeal cancer cell line FaDu were analyzed using flow cytometry. To investigate the biological significance of the CD44+ population, we sorted the CD44+ and CD44– cell populations by using magnetic-associated cell sorting (MACS) technology. After the separation, the purity of the CD44+ cells was determined using flow cytometry. The MTT method was used to detect the different proliferation capabilities of the CD44+ and CD44– cells in vitro. The tumorigenicity of the CD44+ and CD44– cells was determined by injecting CD44+ or CD44– cells (1 × 106 and 1 × 105) into the body of NOD/SCID mice. Results: Some (21.1 ± 1.56)% of the hypopharyngeal cancer cell line FaDu cells expressed CD44. The CD44+ population was efficiently sorted by MACS, and after separation, the purity of the CD44+ cells was (99.4 ± 0.29)%. The MTT assay indicated that the sorted CD44+ cells had a stronger proliferative capacity than the CD44– cells. The tumorigenicity study showed that all the mice injected with 1 × 106 CD44+ cells developed tumors (8/8), half the mice injected with 1 × 106 CD44– cells developed tumors (4/8), 1 of the 8 mice injected with 1 × 105 CD44+ cells developed tumors (12.5%), but none of the mice injected with 1 × 105 CD44– cells developed any tumors (0/8). At the same concentration, the difference in tumorigenic rates between the CD44+ and CD44– groups was statistically significant (Fisher's exact test, p < 0.05). Furthermore, the CD44+ group had a shorter incubation period than the CD44– group. In addition, the average tumor volume of the CD44+ group was (2017.81 ± 538.50) mm3; however, the average tumor volume of the CD44– group was (1153.25 ± 503.18) mm3. The difference was statistically significant (t = 2.67, p < 0.05).

Outer Segment Formation of Transplanted Photoreceptor Precursor Cells

Transplantation of photoreceptor precursor cells (PPCs) into the retina represents a promising treatment for cell replacement in blinding diseases characterized by photoreceptor loss. In preclinical studies, we and others demonstrated that grafted PPCs integrate into the host outer nuclear layer (ONL) and develop into mature photoreceptors. However, a key feature of light detecting photoreceptors, the outer segment (OS) with natively aligned disc membrane staples, has not been studied in detail following transplantation. Therefore, we used as donor cells PPCs isolated from neonatal double transgenic reporter mice in which OSs are selectively labeled by green fluorescent protein while cell bodies are highlighted by red fluorescent protein. PPCs were enriched using CD73-based magnetic associated cell sorting and subsequently transplanted into either adult wild-type or a model of autosomal-dominant retinal degeneration mice. Three weeks post-transplantation, donor photoreceptors were identified based on fluorescent-reporter expression and OS formation was monitored at light and electron microscopy levels. Donor cells that properly integrated into the host wild-type retina developed OSs with the formation of a connecting cilium and well-aligned disc membrane staples similar to the surrounding native cells of the host. Surprisingly, the majority of not-integrated PPCs that remained in the sub-retinal space also generated native-like OSs in wild-type mice and those affected by retinal degeneration. Moreover, they showed an improved photoreceptor maturation and OS formation by comparison to donor cells located on the vitreous side suggesting that environmental cues influence the PPC differentiation and maturation. We conclude that transplanted PPCs, whether integrated or not into the host ONL, are able to generate the cellular structure for effective light detection, a phenomenon observed in wild-type as well as in degenerated retinas. Given that patients suffering from retinitis pigmentosa lose almost all photoreceptors, our findings are of utmost importance for the development of cell-based therapies.

Molecular signature of cancer stem cells isolated from prostate carcinoma and expression of stem markers in different Gleason grades and metastasis

Prostate cancer (PCa) is the most frequently diagnosed malignancy in men worldwide. Chemotherapy response is very poor and resistance to hormone-based treatments is frequent in advances stages. Recently, tumor-initiating cells or cancer stem cells (CSCs) have been identified in several cancers, including PCa. These cells are thought to be responsible for therapy resistance, relapse and metastasis. In the present work, enriched populations of CSCs were obtained using a mixed procedure that included differential clone-forming ability, sphere growing induction (prostatospheres) and magnetic-associated cell sorting (MACS). Also, stem marker expression was determined in PCa biopsies of different histological grades and metastasis samples. The signature for stem markers of the isolated CSCs was CD133+/CD44+/ABCG2+/ CD24-. Expression of stem markers (CD133, CD44, and ABCG2) was higher in medium Gleason biopsies than in lower and higher grades, and lymph-node and bone metastasis samples. These results suggest that the CSCs in PCa reach an important number in medium Gleason grades, when the tumor is still confined into the gland. At this stage, the surgical treatment is usually with curative intention. However, an important percentage of patients relapse after treatment. Number and signature of CSCs may be a prognosis factor for PCa recurrence.

Increased Integration of Transplanted CD73-Positive Photoreceptor Precursors into Adult Mouse Retina

PURPOSE. Retinal degeneration initiated by loss of photoreceptors is the prevalent cause of visual impairment and blindness in industrialized countries. Transplantation of photoreceptor cells represents a possible replacement strategy. This study determined that identification of cell surface antigens can assist in enriching photoreceptor precursors for transplantation. METHODS. The expression profile of rod photoreceptors at postnatal day 4 was investigated by microarray analysis to identify photoreceptor-specific cell surface antigens. For enrichment of transplantable photoreceptors, neonatal retinas from rod photoreceptor-specific reporter mice were dissociated, and the rods were purified by magnetic associated cell sorting (MACS) with CD73 antibodies. MAC-sorted cell fractions were transplanted into the subretinal space of adult wild-type mice. The number of rod photoreceptors contained in unsorted, CD73-negative, and CD73-positive cell fractions were quantified in vitro and after grafting in vivo. RESULTS. Microarray analysis revealed that CD73 is a marker for rod photoreceptors. CD73-based MACS resulted in enrichment of rods to 87%. Furthermore, in comparison with unsorted cell fractions, CD73-positive MAC-sorted cells showed an approximately three-fold increase in the number of integrated, outer segment-forming photoreceptors after transplantation. CONCLUSIONS. CD73-based MACS is a reliable method for the enrichment of integrating photoreceptors. Purification via cell surface markers represents a new tool for the separation of transplantable photoreceptor precursors from a heterogeneous cell population, avoiding the need of reporter gene expression in target cells.

Amniotic-Fluid Stem Cells: Growth Dynamics and Differentiation Potential after a CD-117-Based Selection Procedure

Amniotic fluid (AF) has become an interesting source of fetal stem cells. However, AF contains heterogeneous and multiple, partially differentiated cell types. After isolation from the amniotic fluid, cells were characterized regarding their morphology and growth dynamics. They were sorted by magnetic associated cell sorting using the surface marker CD 117. In order to show stem cell characteristics such as pluripotency and to evaluate a possible therapeutic application of these cells, AF fluid-derived stem cells were differentiated along the adipogenic, osteogenic, and chondrogenic as well as the neuronal lineage under hypoxic conditions. Our findings reveal that magnetic associated cell sorting (MACS) does not markedly influence growth characteristics as demonstrated by the generation doubling time. There was, however, an effect regarding an altered adipogenic, osteogenic, and chondrogenic differentiation capacity in the selected cell fraction. In contrast, in the unselected cell population neuronal differentiation is enhanced.

Purification of CD4 + T Cells for Adoptive Immunotherapy after Allogeneic Hematopoietic Stem Cell Transplantation

Donor lymphocyte infusions (DLIs) are used for adoptive immunotherapy to prevent or treat relapse and infectious complications after allogeneic hematopoietic stem cell transplantation (HSCT). Unmanipulated DLIs are associated with a risk of graft-versus-host disease (GVHD), probably related to CD8(+) T cell activity. We investigated an automated clinical-scale human-CD4(+)-cell purification method to deplete CD8(+) cells. Twenty-four stem cell recipients received a total of 24 leukapheresis products being enriched for CD4(+) cells using magnetic associated cell sorting (MACS) with an automated device (CliniMACS(®)) before DLIs. MACS resulted in a mean CD4(+) cell count of 16 × 10(6)/kg bw corresponding to 3.4-fold CD4(+) cell enrichment. Mean yield and purity of CD45(+)CD3(+)CD4(+)CD14(-)7AAD(-) were 74% ± 23% and 82% ± 11%, respectively. Median initial dose of DLIs was 1.1 × 10(6) CD4(+)/kg. During a median follow-up of 25 months, 7 (30%) patients experienced GVHD (acute II-IV: n = 4, 17%; acute III-IV: n = 2, 8%; chronic limited: n = 2, 8%; chronic extensive: n = 1, 4%). Thirteen of 21 further evaluable patients (62%) showed measurable clinical response, 2 patients with therapy refractory infectious complications (HSV) showed remarkable immunologic improvement. Automated enrichment of CD4(+) by magnetic cell sorting provides an efficient and rapid method for processing donor lymphocytes. Additional studies should further investigate this approach in terms of efficacy and the risk of GVHD.

Mycophenolic Acid Causes Dysfunction of Human Polymorphonuclear Neutrophils and Dendritic Cells during the Interaction with Aspergillus fumigatus.

Immunosuppressive drugs are a variety of substances to prevent transplant rejection. There is increasing data that immunosuppressants not only modulate lymphocyte function but also interfere with other immune effector cells. In this study, we analyzed the influence of Mycophenolic acid (MPA) on the activity of human polymorphonuclear neutrophils (PMNs) and dendritic cells (DCs) during the interaction with the mold Aspergillus fumigatus. PMNs were obtained from blood using Bicoll separation. Release of reactive oxygen species (ROS) was detected photometrically by using dichlorofluorescein. Monocytes were isolated by magnetic-associated cell sorting followed by differentiation into DCs using GM-CSF and IL-4. DCs were either co-cultivated with MPA (10 μg/ml), with A. fumigatus germ tubes (MOI=1) or both stimuli. Whole-genome microarray analyses (Affymetrix U133A) or quantitative real-time PCR assays were performed to quantify differentially regulated genes. MPA could be identified as an enhancer of the A. fumigatus induced oxidative burst of PMNs while fungal killing efficiency was not affected. Incubation of fully differentiated Mo-derived DCs in the presence of MPA did not alter gene expression patterns. However, if DCs were treated during early differentiation processes, DC development was influenced by MPA resulting in increased apoptosis. In addition, we revealed impaired expression of a variety of pro-inflammatory immune response genes (TNF-α, CXCL10, IL-12). In conclusion, MPA influences PMN and DC function during immune defense against fungi. MPA triggers an acute inflammatory syndrome by enhancing the A. fumigatus induced oxidative burst. Furthermore, innate and adaptive immune responses might be impaired because of a reduced number of DCs and lower levels of pro-inflammatory cytokines and chemokines.